CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)‐binding site (CMVpp65mII) is kinase‐deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65‐specific peptides or after infection with either CMV‐Towne strain or rvac‐pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.